RU2118661C1 - Strain of fungus claviceps purpurea (fr) tul - a producer of ergocriptines - Google Patents

Abstract

FIELD: biotechnology, ergot alkaloids. SUBSTANCE: strain Claviceps purpurea (Fr.) Tul VKM F-3602 - a producer of ergocriptines is obtained by method of individual selection from sclerotia of native ergot strain VKM F-2642 D followed by analysis of fungus superelite strains progeny by chemical composition of ergoalkaloids and by the yield. Strain shows 3-fold increase of ergocriptines from square unit as a result of increased content of the end ergoalkaloids in raw and high yield of sclerotia. Ergocriptine yield in biotechnological system fungus-plant is an average 4.8 kg/ha. EFFECT: fungus strain indicated above, increased yield of the end product. 1 tbl

Description

 The invention relates to biotechnology, in particular to the creation of an improved strain of ergocriptine ergot, sclerotia of which is promising as a raw material for the production of ergocriptine alkaloids.

 The proposed strain of Claviceps purpurea BKM F-3602 compared with the prototype (a.a. RF N. 1551731 "Ergot strain BKM F-2642D - producer of ergocriptine alkaloids" / Shain S. S., Voloshina D. A., Kolesnikova L. I. . and others - stated on 02/12/86) provides an increase in the yield of ergocriptins from a unit area of rye infected by the spores of this strain in a known manner due to the high content of ergocriptins in sclerotia and the sum of ergoalkaloids, and a more significant level of raw material productivity.

 The objective of the invention is the selection of an improved strain of ergot Claviceps purpurea (Fr.) Tul. with increased productivity of biologically active alkaloids: alpha and beta ergocriptins.

 The aim of this invention was the creation of a new domestic highly productive ergot strain with good ability to form viable and virulent conidiospores, which, when parasitized in winter rye, would give an increased yield of ergocriptins from artificially infected plant fungi conidiospores due to the accumulation of a high content of ergocriptins with a minimal amount of concomitant algae sclerotia and a large crop of raw materials (horns).

 The above task and goal were achieved thanks to the selected ergocryptin ergot strain, registered June 13, 1996 under the number BKM F-3602 in the All-Russian Collection of Microorganisms at the Institute of Biochemistry and Physiology of Microorganisms of the Russian Academy of Sciences.

 Alkaloids alpha and beta ergocriptines are known medicinal substances that are currently used abroad in medicine in the form of complex preparations, and also serve to obtain pharmacologically active semi-synthetic derivatives based on them that are widely used in medical practice. In the Russian Federation, these alkaloids are used for the industrial production of the original domestic anti-tumor and neurohormonal drug - abergin.

 The creation of strain BKM F-3602, selected by individual selection from sclerotia of the domestic industrial production ergocriptine strain BKM F-2642D, provided the high-quality raw material for the production of the drug Abergin.

 The quality of sclerotia of the proposed strain BKM F-3602 allows for the industrial processing of raw materials to obtain from them the target product - Abergin in an economical way.

 The main advantage of the strain BKM F-3602 is the high productivity of ergocriptins due to the high content of target substances in the raw materials and their predominance in the amount of ergot alkaloids in combination with high yields of sclerotia.

 Thus, a new domestic ergocriptine strain Claviceps purpurea was created, which provides a mushroom – plant plant in the biotechnological system with an increased (by an average of 3.3 times) harvest of ergocriptine alkaloids from a unit of rye cultivation to ergot.

Cultural and morphological features
When saprophytic cultivation of strain BKM F-3602 on a medium from steamed rye grain on the 6-7th day, a white mycelium of the fungus appears, which gradually grows on the surface of the grains. After 15 - 20 days, the mycelium passes into the phase of conidial sporulation, acquiring a grayish tint.

 When the strain is cultivated on an agarized beer wort, the growth of mycelium becomes noticeable 7-10 days after sowing. At the same time, on the surface of the medium, first a white and then a grayish cotton-like mycelium is formed, which fuses tightly with the medium. After 18 - 23 days, conidiophores form and the stage of sporulation begins.

When growing a mushroom on T-2 medium, the following composition (g / l): sucrose 100; asparagine 10; yeast extract 0.1; KCl 0.12; MgSO ₄ • 7H ₂ O 0.25; FeSO ₄ • 7H ₂ O 0.02; ZnSO ₄ • 7H ₂ O 0.015; Ca (NO ₃ ) ₂ 1.0; KH ₂ PO ₄ 0.25; agar agar 15; distilled water 1000 ml.

 Mycelium appears on the 5-6th day after sowing and a staining of the nutrient medium in purple is observed. In the future, the mycelium of the fungus acquires a purple hue.

 The proposed strain on the studied media is characterized by a rather abundant stage of conidial sporulation.

 The sizes of conidiospores during saprophytic cultivation of the strain on these nutrient media are the same and are (3.4 - 8.8) and (2.5 - 5.3) microns.

Physiological signs
The strain Claviceps purpurea BKM F-3602 is a strict aerobic, for which the optimum temperature for its growth and maturation is 24 + 1 ^o C. Claviceps purpurea BKM F-3602, obtained by saprophytic cultivation of the fungus in a nutrient medium from steamed rye grains.

 In 8 - 14 (depending on weather conditions) days after infection in the infected rye flowers, the sphacelial stage of the fungus passes, accompanied by the formation of conidiospores, which are released on the surface of the ears in the composition of the so-called "honey dew". A new generation of conidia provides fungus infection of other flowering plants on the plantation. With extended rye flowering, epiphytotia on the plantation can develop due to subsequent generations of conidiospores in flowers spontaneously infected with the fungus. After 40 - 45 days from the moment of artificial infection of rye, sclerotia (horns) are formed on the ears, containing a significant amount of ergoalkaloids with a predominance of ergocriptins in it.

 Mature sclerotia of the improved ergocriptine ergot strain is oblong, almost trihedral, somewhat curved, tapering to both ends, usually with three longitudinal grooves, black or brownish-violet, 5 to 50 mm long, 1.5 to 8.0 mm thick; the average weight of the horns is 95 mg.

The new strain BKM F-3602 is inoculated under aseptic conditions a sterile medium of steamed rye grain in mattress flasks with a capacity of 1.5 - 2.0 liters. The mushroom crops are thermostated at 24 + 1 ^o C for 30 days, then the flasks are transferred to a dry, darkened room with a temperature of 7 - 9 ^o C and stored until infection with rye, but not more than 2 months. Infection with rye fungus is carried out in the phase of the beginning of heading of a plant by a known technology.

 To this end, an infectious material (an aqueous suspension of conidiospores) is prepared immediately before infection by washing the spores of the fungus with mycelium using tap water on steamed rye grains and then filtering the resulting suspension through a layer of gauze.

The concentration of spores in the infectious fluid should be at least 5 thousand in 1 mm ³ .

 The amount of conidiospores in one flask-mattress is enough to prepare about 6 liters of infectious fluid.

A suspension of conidiospores in water is used no later than 24 hours after its preparation, pre-stirring the liquid. Winter rye infection was carried out on experimental isolated plots with an area of 10 m ² in four biological repetitions manually using needle plates moistened with a suspension of fungal conidiospores. On the infection of one plot consume infectious material on average from two flask mattresses.

 During the growing season, observations were made on the development and growth of ergot in rye (the timing of the appearance of "honey dew" and the nature of flowering plants were taken into account), and the degree of infection of ears and plants was determined. After 6 weeks from the time of rye infection, ergot sclerotia was manually collected. The horns were dried in air in a shaded room to an air-dry state, when the horns became brittle at break.

 The content of the sum of ergoalkaloids and its component composition were determined by known analysis methods. The content of ergocriptins and their accompanying alkaloids in the feed was controlled by the chromato-fluorimetric method, as well as by HPLC.

 The results of biological productivity of sclerotia according to the results of three-year field trials of the proposed ergot strain are presented in the table.

 For the period from 1994 to 1996. yields of sclerotia averaged 682.7 kg / ha with a content of 0.81% of ergoalkaloids and 0.70% of ergocriptins in raw materials. The yield of the target product, ergocriptine alkaloids per 1 ha, averaged 4.80 kg over three years.

Claims (1)

 The strain of the fungus Claviceps purpurea (Fr.) Tub. VKM F-3602 - producer of ergocriptins.

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